Intranasal Exosomes for Treatment of Neuroinflammation?: Prospects and Limitations

Brain inflammatory diseases such as multiple sclerosis, acute disseminated encephalomyelitis, viral encephalitis, and bacterial meningitis, as well as other central nervous system conditions with an inflammatory component (e.g., schizophrenia, migraine headaches, and neurodegenerative disorders such as Parkinson's and Alzheimer's diseases), are the subject of extensive translational research to develop therapies addressing the underlying inflammation. Microglial cells—the brain's resident macrophages—have been shown to contribute significantly to inflammation in these diseases by producing the inflammatory interleukins IL-1 and IL-6 and are therefore ideal targets for anti-inflammatory therapies. Unfortunately, there is a lack of suitable delivery strategies that specifically target brain microglial cells. However, in this issue of Molecular Therapy, Zhuang et al. describe the use of exosomes to deliver anti-inflammatory drugs to the brain through a non­invasive intranasal route.¹ The therapeutic value of this approach was demonstrated with exosome-complexed curcumin in lipopolysaccharide (LPS)-induced inflammation and experimental allergic encephalomyelitis and with an exosome-complexed Stat3 (signal transducer and activator of transcription 3) inhibitor in a glioblastoma tumor model. Although the efficacy of curcumin and Stat3-targeting inhibitors have been validated extensively in a large number of inflammatory neurological diseases, their poor bioavailability and inability to cross the blood–brain barrier (BBB) have presented obstacles to their clinical use. The new findings demonstrate that exosomes administered intranasally are potential delivery vehicles for these therapeutic agents, by increasing their biological stability and enabling them to bypass the BBB. However, the mechanism of exosome translocation into the brain from the nasal cavity and the applicability of this approach to higher-molecular-weight therapeutic agents need to be elucidated before the therapeutic potential of intranasal exosome delivery in the clinical setting can be fully assessed.

Exosomes are naturally occurring membranous nanovesicles 40 to 100 nm in diameter. They arise from the endocytic cellular pathway through inward budding of the limiting late endosomal membrane, giving rise to multivesicular bodies, which then fuse with the plasma membrane to release their vesicular contents (exosomes). A breakthrough in the understanding of the biological significance of exosomes came from proteomic and transcriptomic profiling studies showing that exosomes are natural carriers of protein and nucleic acids, including messenger RNA (mRNA) and microRNA.² Exosomes may have pleiotropic biological functions, including regulation of vascular homeostasis, antigen presentation to T cells, angiogenesis, cytokine transport, transmission of reactive oxygen species, and transfer of mRNA for de novo translation in the recipient cell.^3,4,5 Furthermore, they have been implicated in the progression of disease processes by spreading oncogenes, infectious particles, and neuropathogenic proteins between cells.^6,7 Some types of exosomes have been shown to possess intrinsic therapeutic value; for example, exosomes from mesenchymal stem cells possess cardioprotective properties in mouse models of ischemia reperfusion injury.⁸ More recently, research in our laboratory provided the first demonstration of the potential of exosomes as targeted vehicles of therapeutic oligonucleotide delivery to the brain, paving the way for the exploitation of exosomes in the delivery of other macromolecular drugs in vivo.⁹

Zhuang et al.¹ build on previous findings by the same researchers that T-cell-derived exosomes are preferentially taken up by immature myeloid cells such as macrophages and microglial cells.¹⁰ Given the selective uptake of exosomes by macrophages, already demonstrated by these authors, and the newly established properties of exosomes as drug delivery vehicles,⁸ Zhuang and colleagues investigated whether T-cell-derived exosomes could be used to deliver anti-inflammatory agents specifically to microglial cells in the brain. They used a noninvasive intranasal route to bypass the BBB. Unloaded exosomes derived from a variety of mouse cell lines and labeled with a biological dye were first administered intranasally 2 µg at a time over a 10-minute period. Labeled exosomes were detected in the olfactory bulb within 30 minutes and up to 24 hours after administration. Multiorgan imaging demonstrated their selective homing to the brain and, to a lesser extent, the intestine. These initial experiments with unloaded exosomes demonstrated their fast and selective homing to the brain after intranasal administration.

Zhuang and co-workers subsequently examined the ability of exosomes to deliver the anti-inflammatory molecule curcumin to the brain. Curcumin was complexed with exosomes (Exo-cur) by incubation at room temperature, and after delivery its concentration in brain lysates was measured using high-pressure liquid chromatography. Curcumin was detected in brain lysates within 1 hour of Exo-cur administration and thereafter for up to 12 hours. By counter­staining brains from treated mice with the microglial marker Iba1, the authors established that most microglial cells contained exosomes within 1 hour of delivery, and they concluded that these cells were preferentially targeted by exosomes.

To explore the potential of intranasally administered exosomes as delivery vehicles for the treatment of neuroinflammatory diseases, the authors used three models of brain inflammation. First, in a model of LPS-induced inflammation, Exo-cur led to a reduction in the number of activated inflammatory microglial cells (CD45.2⁺, IL-B⁺) in the brains of LPS-challenged mice within 2 hours of administration, when compared with control empty-exosome treatment. The observed reduction in activated microglial cells was attributed to apoptosis, which in turn correlated positively with the levels of curcumin delivered to the brain. Second, in a model of myelin oligodendrocyte glycoprotein–induced experimental allergic encephalomyelitis, mice that received Exo-cur daily for 31 days had significantly reduced disease severity when compared with empty-exosome or curcumin-alone treatments. This effect was also associated with a decrease in IL-1β expression in micro­glial cells. Third, in a glioblastoma model, tumors were established by injecting GL26 tumor cells expressing luciferase intracranially, and mice were then treated with exosome-encapsulated Stat3 inhibitor for 12 consecutive days beginning on day 3. Tumor growth, assessed by luciferase activity, was significantly delayed by the treatment, and mouse survival was prolonged as compared with control treatments. This effect was associated with a reduction in Stat3 activation in microglial cells, suggesting that the Stat3 inhibitor was successfully delivered to the brains of these mice.

Zhuang and colleagues' study represents further proof of concept for exosomes as drug delivery vehicles, with fast and selective homing to the brain. This was validated in three neuroinflammation disease models and supported by experimental evidence of the mechanism of action of these exosome-delivered drugs. Moreover, the study builds on our earlier findings⁹ by demonstrating a greater potential of exosomes beyond oligo­nucleotide delivery so as to include a potentially wider range of cargoes. This warrants further exploration of other types of biological cargo—most notably proteins and antibodies, and perhaps even agents such as oncolytic viruses. Although it was established in our laboratory that exosomes targeted to acetylcholine receptor cross the BBB when administered intravenously, it is nonetheless exciting that the current study proposes the intranasal route as an additional noninvasive route for brain delivery.

Zhuang et al.¹ demonstrate targeting of brain microglial cells by exosomes but do not address the mechanism of this selectivity. Although exosomes are generally thought to be taken up into recipient cells through the endosomal pathway,¹¹ it is not clear whether T-cell-derived exosomes utilize the endosomal pathway for entry in microglial cells and whether there are properties specific to microglial cells (e.g., receptor-mediated uptake) that confer the reported selectivity. It would also be of interest to determine the extent of exosome homing to the various parts of the brain and spinal cord, and whether other glial cell populations such as astrocytes are targeted by exosomes. In addition, it is unclear whether exosome uptake by microglial cells in the brain is dependent on or enhanced by concomitant inflammation. Addressing these questions will help assess the applicability of this technology to noninflammatory neurological diseases.

The mechanism of exosome trans­location from the nasal cavity to the brain is not addressed in this study. Whereas the observed kinetics of exosome translocation to the brain are most consistent with the trigeminal and the olfactory routes proposed by Thorne et al.,¹² further studies are warranted to pinpoint the route by which exosomes travel to the olfactory bulb and ultimately throughout the nervous system, and to identify the parameters that control the efficacy of brain homing, so as to refine the quality of exosomes for this purpose. In addition, the study uses exosomes derived from cell lines as opposed to primary cells. Given that cell lines are often immortalized by stable expression of oncogenes, along with the reported role of exosomes in the transport of pathogenic proteins between cells,^6,7 it would be important to characterize the endogenous cargoes of cell line–derived exosomes and to investigate the long-term effects of their use in vivo. Finally, the study demonstrates an association between exosomes and curcumin following coincubation, but it does not formally distinguish between the possibilities that curcumin is encapsulated within these exosomes and that it is associated with the exosomal membrane. To distinguish between these two scenarios is crucial; the difference has bearing on the future use of exosomes with other drug cargoes. Therefore, detailed structural studies are warranted to determine the nature of the association between exosomes and curcumin.

In summary, the study by Zhuang et al.¹ represents further validation of the potential of exosomes as drug delivery vehicles and demonstrates their versatility in terms of both delivery route and cargo type. Demonstration that exosomes have natural selective properties for certain target cells makes them even more attractive as drug delivery vehicles with higher specificity and fewer side effects. Although the route of exosome homing to the brain from the nasal cavity remains unknown, as does the nature of the association between exosome and cargo, there is little doubt that exosomes hold great promise for the field of drug delivery to the brain and across otherwise impermeable biological barriers.